Biology subjects

Hejazi, S. S.

Publications and source records attributed to Hejazi, S. S..

3 recordsLinked to original sources

bioRxiv2026

High-Throughput FRET Affinity Screening Technique (HTFAST) For Cell-Free Expressed Binding Protein Characterization

The rapid engineering of high-affinity binding proteins, such as nanobodies and single-domain antibodies (sdAbs), is increasingly driven by cell-free, machine-learning-guided optimization. However, high-throughput, quantitative characterization of binding affinity remains a major bottleneck, particularly for proteins expressed in cell-free systems without purification. Here, we present High-Throughput FRET Affinity Screening Technique (HTFAST) for rapid affinity characterization of binders expressed directly in crude E. coli cell-free protein synthesis reactions. HTFAST leverages Forster resonance energy transfer (FRET) between fluorescent-protein-fused binders and dye-labeled antigens to enable real-time, quantitative measurement of equilibrium dissociation constants. We systematically optimized fluorophore pairs used and labeling parameters using the SpyTag003-SpyCatcher003 model system. Using donor-quenching and acceptor-emission FRET analyses, HTFAST reliably quantified nanomolar binding affinities in crude lysates for SpyTag003-SpyCatcher003 model system. We validated the platform for nanobodies by characterizing a CD4-binding nanobody, Nb457, and benchmarking multiple SARS-CoV-2 receptor-binding domain sdAbs, demonstrating HTFASTs ability to rank binding strengths across a range of affinities. Finally, we demonstrate that both binding partners can be expressed directly in CFPS, further streamlining screening workflows. Overall, HTFAST provides a scalable, quantitative, and cell-free-compatible approach for high-throughput affinity screening, well suited for DBTL campaigns aimed at accelerating the development of next-generation binding proteins.

bioengineering↗

bioRxiv2025

Rapid Cell-Free Combinatorial Mutagenesis Workflow Using Small Oligos Suitable for High-Iteration, Active Learning-Guided Protein Engineering

Active learning-guided protein engineering e2iciently navigates the challenging fitness landscape by screening designs iteratively in a model-guided design-build-test-learn cycle. However, while high iterations boost performance, current workflows reliance on tedious and costly cell-based cloning and expression steps limits the iterations they can practically implement. To address this problem, we present a novel combinatorial mutagenesis workflow that uses small ([~]20-40 bp) mutagenic annealed-oligo fragments and cell-free expression to rapidly and conveniently screen protein variants in <9 hours. Using bulk-prepared mutagenic oligos eliminates the need for cloning, PCR-based mutagenesis, or ordering costly genes each screening round. Their >80% size reduction from current fragment-based shu2ling strategies also helps avoid including multiple mutations on the same fragment, reducing the number one must order to cover the design space. By screening 3-10 fragment assemblies for two di2erent proteins, we show our approach is a general, scalable, and cost-e2ective platform for high-iteration protein engineering.

bioengineering↗

bioRxiv2024

Amplified DNA Heterogeneity Assessment with Oxford Nanopore Sequencing Applied to Cell Free Expression Templates

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

bioinformatics↗